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The Journal of Immunology, 1999, 162: 5263-5269.
Copyright © 1999 by The American Association of Immunologists

Expression of L-Selectin Ligands by Transformed Endothelial Cells Enhances T Cell-Mediated Rejection1

Luigi Biancone*, Ivan Stamenkovic{dagger}, Vincenzo Cantaluppi*, Mariarosaria Boccellino*, Antonella De Martino*, Federico Bussolino{ddagger} and Giovanni Camussi2,*

* Chair of Nephrology, Department of Internal Medicine, University of Torino, Torino, Italy; {dagger} Department of Pathology, Harvard Medical School and Pathology Research, Massachusetts General Hospital, Charlestown, MA 02129; and {ddagger} Department of Genetics, Biology, and Biochemistry, University of Torino, and Institute for Cancer Research and Treatment, Candiolo, Italy


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Recent immunohistochemical studies have suggested that L-selectin ligands may be implicated in the infiltration of tumors and rejected transplants by lymphocytes. In the present study, polyoma-middle T Ag-transformed endothelial cells (H.end), which typically form in vivo immunogenic vascular tumors resembling Kaposi’s sarcoma, were engineered to express L-selectin ligands by stable transfection with a cDNA encoding {alpha}(1,3/4)-fucosyltransferase (H.endft). The ability of these cells to form tumors in the s.c. tissues of normal and immunocompromised mice was then compared with that of H.end cells transfected with the hygromycin-resistance vector only (H.endhygro). H.endhygro cells rapidly formed local and metastatic tumors in normal syngeneic mice, leading to death within 2–3 mo postinjection. By contrast, tumors derived from H.endft cells displayed a slower rate of growth, an absence of metastasis, and marked lymphocyte infiltration. Animals bearing these tumors survived for a significantly longer duration than animals injected with H.endhygro cells. Alternatively, H.endft and H.endhygro cells formed tumors with comparable aggressiveness in immunocompromised mice, resulting in animal death within 3 wk of injection. H.endft but not H.endhygro cells supported L-selectin-dependent adhesion and cytolytic T cell activity in vitro. Taken together, our observations indicate that the in situ expression of fucosyltransferase may significantly influence the cellular immune response in endothelioma tumors. These results may be relevant in understanding the development of vascular opportunistic tumors such as Kaposi’s sarcoma.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Lymphocyte trafficking from the circulation to lymphoid organs or extralymphoid tissues is a multistep process requiring a series of sequential interactions between specific adhesion receptor-ligand pairs (1). A critical role in the initial events of this process is played by L-selectin, a member of the selectin family of adhesion receptors that is constitutively expressed on most leukocytes (2, 3). Interaction between leukocyte L-selectin and endothelial cell ligands, which are composed of appropriately presented sialylated, fucosylated, and/or sulfated oligosaccharides (4), promotes leukocyte rolling on the endothelial surface facilitating subsequent leukocyte arrest and extravasation (2, 3, 4). Several endothelial glycoprotein ligands of L-selectin have been identified, including mucin-like proteins GlyCAM-1 and CD34, Sgp200, and a subset of MAdCAM-1 molecules (4). Appropriate glycosylation of these protein cores is indispensable for recognition by L-selectin to occur (5). The importance of appropriate L-selectin function has been underscored by observations that L-selectin-deficient mice display major defects in lymphocyte homing and leukocyte rolling on endothelium (6). Leukocyte rolling and extravasation are also severely impaired in P-selectin-deficient mice (7), whereas E-selectin-deficient mice have no obvious changes in leukocyte trafficking during an inflammatory response (8). L-selectin counterreceptors are physiologically expressed by specialized endothelial cells termed high endothelial venules lining the postcapillary venules of peripheral lymph nodes (4). However, similar high endothelial venule structures that express L-selectin ligands can be observed at sites of chronic inflammation where L-selectin also appears to play an important role in mediating lymphocyte recruitment (9). In addition, immunohistochemical studies have recently suggested a correlation between the expression of L-selectin ligands by tumor vessels and lymphocyte infiltration (10) as well as in rejected transplants (11). Furthermore, fucosyltransferase (ft)3-dependent expression of L-selectin ligands was detected in several tumor cell lines (12). Taken together, these data suggest that the expression of functional L-selectin ligands may facilitate lymphocyte infiltration and a local cellular immune reaction in pathological events such as an antitumor immune response or transplant rejection. However, direct evidence of the role of these ligands in these conditions is lacking to date.

To address the possible role of the expression of fucosylated oligosaccharides in tumor rejection in vivo, {alpha}(1,3/4)-ft-specific cDNA (13), was stably transfected into a polyoma-middle T oncogene-transformed endothelial cell line, H.end, that lacks L-selectin ligands and typically forms an immunogenic vascular tumor when injected in vivo in syngeneic mice. This tumor has been used previously as a murine model for Kaposi’s sarcoma (14, 15). Previous studies have demonstrated that transfection of {alpha}(1,3/4)-ft does not modify the growth of a nonimmunogenic melanoma tumor per se (16). The aim of the present study was to evaluate whether the expression of fucosylated ligands by an immunogenic tumor potentiates the effector phase of rejection as a result of adhesion molecule-mediated lymphocyte recruitment. Therefore, growth and metastatic dissemination of {alpha}(1,3/4)-ft and control H.end transfectants were compared in normal and immunocompromised mice. The results obtained indicate that the expression of {alpha}(1,3/4)-ft in H.end cells promotes an L-selectin-dependent recruitment of lymphocytes toward the tumor and enhances tumor rejection. This effect was absent in immunocompromised mice.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell lines and transfectants

Murine H.end endothelioma cells (17, 18) were cultured in DMEM (Irvine Scientific, Santa Ana, CA) supplemented with 2 mM glutamine (Life Technologies, Gaithersburg, MD), 10% FCS (Irvine Scientific), and gentamicin. Cells were transfected with {pi}H3M vector containing the hygromycin resistance gene only or with {pi}H3M vector containing {alpha}(1,3/4)-ft cDNA (13). Transfectants were generated by electroporation (Gene Pulser; Bio-Rad, Richmond, CA) at 250 V and 960 µF in 4-mm electroporation cuvettes. Clones were selected for hygromycin resistance in DMEM, 10% FCS, and 500 µg/ml hygromycin B (Boehringer Mannheim, Indianapolis, IN) and were tested for sialyl Lewis a (SLea) expression by indirect immunofluorescence.

Determination of cellular growth rate in vitro

Each transfectant was cultured in 96-well, flat-bottom microtiter plates (Falcon Labware, Oxnard, CA) at a concentration of 5 x 104 cells/well in DMEM/10% FCS. After 24 h of culture, cells were washed and incubated in serum-free DMEM containing 250 µg/ml 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide at 37°C. Cell growth was monitored by determination of the absorption values at 620 nm in an automated ELISA reader. All cultures were done in triplicate.

Immunofluorescence studies

For cytofluorometric analysis, cells were detached from plates with EDTA, washed, resuspended in PBS, and incubated at 4°C for 30 min with RPMI 1640 containing 10 µg/ml human L-selectin-Ig fusion protein (L-selectin receptor globulin (Rg)) (19); human recombinant CD8-Ig fusion protein (CD8 Rg), which was shown previously to be nonreactive with murine tissues (20), was used as a control. As a second-step reagent, FITC-conjugated anti-human IgG (Sigma, St. Louis, MO) was used. Cells were analyzed on a FACS (Becton Dickinson, Mountain View, CA).

For tissue staining, 5-µm paraffin-embedded tissue sections were stained with 10 µg/ml of goat anti-mouse CD4 or rabbit anti-mouse CD8 (Santa Cruz Biotechnology, CA) or with control isotype-matched Ab (PharMingen, San Diego, CA) for 45 min at room temperature. The slides were washed in PBS, incubated with fluorescein-labeled rabbit anti-goat or goat anti-rabbit IgG affinity-purified Ab (Sigma) for 30 min at room temperature, washed, counterstained with 1 µg/ml propidium iodide (PI) in PBS for 30 s, mounted with anti-fade mounting medium (Vector Laboratories, Burlingame, CA), and examined.

Adhesion and cell-mediated cytotoxicity assays

Adhesion was studied in nonstatic conditions according to Spertini et al. (21). The Jurkat cell line (American Type Culture Collection, Manassas, VA), which expresses L-selectin (22), was used to test lymphoid cell adhesion to H.end transfectants. Approximately 55 µCi of [111In]oxine was added drop-wise to 2 ml of Jurkat (107) suspension and allowed to incubate at room temperature for 10 min. After centrifugation at 1400 x g for 10 min, cells were resuspended in Tris-buffered Tyrode’s solution containing 0.5% heat stable Ag. The labeling efficacy was ~90%; the viability of Jurkat cells, as determined by trypan blue exclusion, was always >95%. H.end transfectants, which were grown to confluence in 24-well plates, were washed three times with RPMI 1640 medium containing 0.5% heat stable Ag and placed on a platform rotator (80 rpm); next, 111In-labeled Jurkat cells were added to the plate at 2 x 105 cells/well at 4°C for 30 min. After the incubation periods, nonadherent Jurkat cells were removed by washing three times with the incubation medium. The adherent radiolabeled Jurkat cells were then solubilized for 10 min with 1N NaOH and 1% SDS, and the lysate radiolabel was determined in a gamma counter. In some experiments, 10 mM EDTA was added to the wells or Jurkat cells were preincubated for 10 min at 4°C with 20 µg/ml anti-L-selectin blocking mAb (clone DREG-56; PharMingen) before the coincubation. The cell-mediated cytotoxicity assay has been described by Kroesen et al. (23). Briefly, mice were injected s.c. with H.end cells, and splenocytes were harvested on day 15. H.endft or H.endhygro were labeled overnight with 3,3'-dioctadecylloxacarbocyanine (Molecular Probes, Eugene, OR), washed, and incubated at 37°C for 24 h with the harvested splenocytes at different E:T ratios. At the end of the incubation, a 3.75-mM solution of the membrane-impermeant nucleic acid counterstain PI was added to label any cells with compromised plasma membrane, and cells are observed under a fluorescence microscope. The percentage of dead target cells in the presence of effector cells (+effectors) corrected for spontaneous target cell death in the absence of effector cells (-effectors) was calculated according to the following equation, where G = green and G+R = both green and red. The corrected percentage of cytotoxicity is equal to: ([(G+R cells/G cells) (+effectors)] - [(G+R cells/G cells) (-effectors)]) x 100. In selected experiments, 20 µg/ml anti-mouse L-selectin blocking mAb (clone MEL-14, PharMingen) or isotype-matched control IgG (PharMingen) were added to the cells.

Evaluation of tumor growth in vivo

For in vivo experiments, cells were gently detached from plates with EDTA, washed with PBS, counted in a microcytometer chamber, and resuspended in saline. A total of 107 cells, which is the minimal tumorigenic dose of tumor cells required for outgrowth in 100% of syngeneic mice (15), in a total volume of 150 µl were injected s.c. into the left back of mice via a 26-gauge needle and using a 1-ml syringe. The mice were scored for tumor growth once a week, and tumor size was documented by measuring two perpendicular diameters in millimeters using a caliper. Animals were sacrificed at different timepoints and subjected to autopsy. All organs were examined macroscopically for evidence of tumor growth. Portions of liver, annexes, lung, and brain tissue from each animal as well as any tissue containing visible tumor growth were fixed in formaldehyde for light microscopy and immunohistochemical studies.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
H.end cells implanted in syngeneic mice typically form tumors similar to hemangiosarcomas and Kaposi’s sarcoma characterized by lymphocyte infiltration (14, 15, 24). It has been shown that these tumors induce a T cell-dependent antitumor immune response and may therefore represent a suitable model for opportunistic vascular neoplasias (15). H.end cells do not express fucosylated L-selectin ligands, as shown by the absence of binding to soluble L-selectin (Fig. 1Go). H.endft variant cells were developed that stably express a cDNA encoding an {alpha}(1,3/4)-ft, which catalyzes transglycosylation reactions yielding both Fuc{alpha}(1, 3)- and Fuc{alpha}(1, 4)-glycosidic bonds (13) and directs the expression of L-selectin ligands (14). H.endft but not H.end cells transfected with the hygromycin resistance selection vector only (H.endhygro) were observed to bind L-selectin-Ig fusion protein (L-selectin Rg) (Fig. 1Go). In addition, H.endft cells supported a 4-fold increase in Jurkat cell adhesion compared with H.endhygro cells (Fig. 2Go). The enhanced Jurkat adhesion was L-selectin- and Ca2+-dependent, as shown by the inhibitory effect of anti-L-selectin mAb and EDTA, respectively (Fig. 2Go). In vitro proliferation assays demonstrated a comparable growth of H.endft and H.endhygro cells (data not shown). To evaluate whether the expression of ft by target cells may influence CTL killing, we studied the in vitro cytolytic effect on H.endft and H.endhygro cells of splenocytes from both mice carrying H.end tumor and naive mice. As shown in Fig. 3GoA, the CTL activity from the T lymphocytes of mice carrying H.end tumors on H.endft cells was significantly increased compared with that on H.endhygro cells. Minimal cytotoxic activity was observed when lymphocytes from naive mice were used instead of cells from mice carrying H.end tumors (Fig. 3GoB). Moreover, the treatment of lymphocytes from mice carrying H.end tumors with anti-CD3 mAb abrogated cytotoxicity, suggesting T cell-mediated CTL killing (Fig. 3GoA). The enhanced cytotoxicity observed with T lymphocytes from mice carrying H.end tumors on H.endft cells was supported by L-selectin-mediated adhesion, because it was inhibited by anti-L-selectin mAb (Fig. 3GoA).



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  		FIGURE 1. Binding of L-selectin Rg on H.endhygro and H.endft cells. CD8 Rg (control), in place of L-selectin Rg, displayed an absence of staining on both H.endhygro and H.endft. The results shown are representative of four separate experiments.

 


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  		FIGURE 2. In vitro adhesion assay of Jurkat cells to H.endhygro and H.endft cells. 111In-labeled Jurkat cells were added to H.endhygro and H.endft cell monolayers at 2 x 105 cells/well. Adhesion assays were performed at 4°C for 30 min on a platform rotator (80 rpm). After the incubation periods, nonadherent Jurkat cells were removed by washing, adherent cells were lysed, and the lysate radiolabel was determined in a gamma counter. In some experiments, 10 mM EDTA was added to the wells, or Jurkat cells were preincubated with anti-L-selectin blocking mAb as described in Materials and Methods. The results shown are representative of three separate experiments. ANOVA with Newman-Keul’s multicomparison test was performed. Statistical differences (p < 0.05) were encoded as follows: H.endhygro alone vs H.endft alone (*), H.endhygro alone vs H.endhygro with EDTA or anti-L-selectin mAb (°), and H.endft alone vs H.endft with EDTA or anti-L-selectin mAb (§).

 


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  		FIGURE 3. Comparison of CTL activity on H.endhygro and H.endft cells. Splenocytes (effector cells) were harvested from mice bearing H.end-derived tumors for 15 days (A) or from naive mice (B) and incubated with H.endhygro or H.endft cells (target cells) at different E:T ratios for 24 h at 37°C. The addition of 20 µg/ml anti-mouse L-selectin blocking mAb (clone MEL-14) significantly affected the lysis of H.endft cells but not of H.endhygro cells. The addition of 5 µg/ml anti-mouse CD3 mAb abrogated the lysis of H.endhygro. The addition of an isotype-matched control IgG gave results that were similar to those for vehicle alone (data not shown). ANOVA with Newman-Keul’s multicomparison test was performed. Statistical differences (p < 0.05) were encoded as follows: H.endhygro alone vs H.endft alone (*), H.endhygro alone vs H.endhygro with anti-L-selectin mAb (°), and H.endft alone vs H.endft with anti-L-selectin mAb (§).

 
To address the effect of ft expression on H.end tumor growth in vivo, an equal number of cells (107) from each transfectant (H.endhygro and H.endft) was first injected s.c. in nude mice (six per group), and the animals were monitored for visible tumor growth. Both H.endhygro and H.endft cells formed massive tumors (Fig. 4Go) within 2 wk, leading to death in 2–3 wk. No significant difference between these two groups of nude mice was found with regard to tumor size (Fig. 4Go) and dissemination (number of metastases at autopsy: H.endhygro, 9 ± 4; H.endft, 12 ± 5), which typically consists of several small metastases in the annexial organs (15). This result suggests that the expression of {alpha}(1,3/4)-ft does not influence tumor growth and dissemination in the absence of an intact immune response.



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  		FIGURE 4. Growth of tumors induced by H.endhygro and H.endft cells in syngeneic DBA/2 and nude mice. Mice were injected s.c. as described in Materials and Methods and sacrificed after 4 wk. All of the nude mice injected with either H.endhygro or H.endft died before the endpoint at 2–3 wk postinjection. Results were expressed as the mean diameter (in millimeters) of tumors from groups of six mice each. ANOVA with Dunnett’s multicomparison test was performed. Statistical differences (p < 0.05) were encoded as follows: H.endhygro in nude mice vs H.endft in nude mice (*) and H.endhygro in DBA/2 mice vs H.endft in DBA/2 mice (§).

 
To address the effect of ft expression on H.end tumor growth in normal animals, H.endhygro and H.endft cells were compared for tumor formation in the s.c. tissues of syngeneic DBA/2 mice (six mice per group). The growth of H.endhygro cell-derived tumors in syngeneic animals was significantly retarded compared with their growth in immunocompromised animals (Fig. 4Go), which is consistent with previous observations (15). At the 4-wk endpoint, all of the mice in this group developed a primary tumor mass (Figs. 4Go and 5Go) with secondary metastasis in the annexial organs (number of metastases: 5 ± 3). As shown in Fig. 5Go, A and B, H.endhygro grew as a hemangiosarcoma with characteristic vascular lacunae containing erythrocytes that strongly resembled Kaposi’s sarcoma. A moderate infiltration of inflammatory cells was detectable within the tumor (Fig. 5GoB). Tumors derived from H.endft cells after an initial growth showed a marked reduction in size with respect to H.endhygro-derived counterparts (Fig. 4Go). At the site of implantation, residual tumors showed a marked infiltration of inflammatory cells surrounding a central necrotic area (Fig. 5Go, C and D). The vascular lacunae were absent or seen occasionally. Moreover, metastases were absent. These results suggest that the expression of {alpha}(1,3/4)-ft conditions the local growth and the metastatic dissemination of H.end tumors in vivo. Similar results were obtained when the in vivo experiments were repeated with two other transfectant isolates of H.endhygro and H.endft cells (three mice per group) to dispel the possibility of a clone-specific effect (data not shown).



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  		FIGURE 5. Histological (hematoxylin and eosin) analysis of tumors from normal DBA/2 mice injected with H.endhygro and H.endft cells and sacrificed after 4 wk. A and B, Micrographs showing the typical aspects of H.endhygro large tumor masses with vascular lacunae containing erythrocytes (A, x100 magnification; B, x400 magnification). C and D, Micrographs showing aspects representative of rejected H.endft tumors (C, x80 magnification; D, x400 magnification). The central necrotic area is surrounded by a marked inflammatory infiltrate. In D, aspects of karyolysis and karyorhexis are seen.

 
Immunohistochemical analysis revealed minimal to moderate infiltration of either CD4+ or CD8+ T cells within H.endhygro tumors (Table IGo and Fig. 6Go, A and B). By contrast, H.endft tumors were massively infiltrated by both CD4+ and CD8+ T lymphocytes (Table IGo and Fig. 6Go, C and D).


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  		Table I. Immunohistochemical analysis of tumor-infiltrating T lymphocytes

 


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  		FIGURE 6. Immunohistochemical analysis of tumors from normal DBA/2 mice injected with H.endhygro and H.endft cells and sacrificed after 4 wk. A and B, Micrographs representative of a minimal to moderate infiltration of CD4+ (A) and CD8+ (B) T lymphocytes in tumors induced by H.endhygro. C and D, Micrographs representative of a massive infiltration of CD4+ (C) and CD8+ (D) T lymphocytes in H.endft tumors. Slides were stained as described in Materials and Methods and counterstained in red with PI (A–D, x400 magnification).

 
To compare the survival rate of mice injected with H.endhygro or H.endft cells, a second set of experiments with an endpoint of 3 mo was performed. The results showed a significant prolongation of survival of mice injected with H.endft compared with mice injected with H.endhygro. At the completion of this study, all of the animals (six of six) injected with H.endhygro cells were dead due to tumor progression, whereas survival was 100% for mice (six of six) injected with H.endhygro cells (Fig. 7Go).



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  		FIGURE 7. Survival of syngeneic DBA/2 and nude mice bearing tumors induced by H.endhygro and H.endft cells. Mice (six per group) were injected s.c. as described in Materials and Methods and monitored for 3 mo. All of the syngeneic DBA/2 mice injected with H.endhygro cells were dead within 11 wk due to tumor progression; in contrast, at the end of the experiments (12 wk), survival was 100% for mice injected with H.endhygro cells. Kaplan-Meier analysis indicates that the survival of DBA/2 mice injected with H.endft cells was significant vs the survival of H.endhygro-injected DBA/2 mice (Log-rank: {chi}2 = 12.0940, p = 0.0005). In contrast, both H.endhygro and H.endft cells injected in nude mice led to death within 3 wk, without a significant difference in survival (Log-rank: {chi}2 = 0.0238, p = 0.8773).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The expression of fucosylated oligosaccharides regulates selectin-mediated leukocyte adhesion to and rolling on lymphoid and inflammatory tissue endothelia and thereby participates in lymphocyte homing to lymphoid tissues and in leukocyte recruitment to sites of injury (3, 4). Recent immunohistochemical studies have suggested that L-selectin ligands may also be implicated in lymphocyte infiltration of tumors and rejected organs (10, 11). However, direct evidence that fucosylated selectin ligands may play a role in tumor rejection is lacking. The results of the present study demonstrate the potential importance of fucosylated L-selectin ligand expression in the efficiency of an immune response to a vascular opportunistic tumor that resembles Kaposi’s sarcoma. Indeed, the in vitro experiments demonstrated that T lymphocytes derived from animals bearing an H.end tumor expressed more efficient cytotoxic activity on cells expressing the fucosylated L-selectin ligands. L-selectin ligand expression on H.end may have potentially contributed to an enhancement of antitumor immunity by at least two mechanisms. First, enhanced physical lymphocyte-tumor cell contact may augment the efficiency for lymphocyte cytotoxicity toward tumor cells. In support of this notion, in vitro adhesion assays showed a marked increase in lymphoid cell attachment to H.end cells expressing ft in comparison with vector only transfectants. The observed adhesion was L-selectin-dependent as demonstrated by its occurrence at 4°C under nonstatic conditions, by EDTA sensitivity, and by abrogation by anti-L-selectin blocking Abs. The second mechanism may possibly rely on the induction of lymphocyte extravasation and recruitment, because H.end cells may themselves line capillary walls as observed previously by Williams et al. (14). A similar situation occurs in Kaposi’s sarcoma, a hemangiosarcoma, for which development is favored by an impairment of immune surveillance. Moreover, the dissimulation of tumor cells into capillary lumen and direct contact with the circulation are common features of several vascular tumors. The expression of fucosylated L-selectin ligands may also favor recruitment of the polymorphonuclear cells that were effectively seen in the inflammatory infiltrate detected within H.endft tumors. However, the experiments performed in nude mice suggest that the T cell-mediated response rather than aspecific leukocyte recruitment is critical for endothelioma rejection.

H.end cells have been shown previously to form tumors that are highly immunogenic (15). In a previous study (16), we transfected the same {alpha}(1,3/4)-ft in B16F10 murine melanoma cells, which are known to be poorly immunogenic (25), and tested for tumorigenicity in syngeneic C57BL/6 mice. Under such conditions, no differences were observed with regard to tumor growth or lymphocyte infiltration that is basely poor or absent. This finding may suggest that the expression of selectin ligands might affect tumor growth only in the presence of an ongoing local antitumor immune response by favoring the recruitment of T lymphocytes. In addition, the endothelial origin of H.end cells has provided a more suitable model to evaluate the effect of ft expression on antitumor immunity.

In conclusion, our study suggests that the expression of L-selectin ligands by endothelial tumor cells enhances cell-mediated immunity and reduces tumor aggressiveness in vivo. The demonstration that local endothelial expression of functional L-selectin ligands facilitates lymphocyte infiltration and a T cell-mediated immune response may also contribute to the elucidation of the mechanisms of other pathological conditions, such as inflammation and transplant rejection, where the detection of L-selectin ligands has been reported recently (26, 11).


   Acknowledgments
 
We thank Dr. R. Bonomini for discussions and helpful suggestions.


   Footnotes
 
1 This work was supported by the Associazione Italiana per la Ricerca sul Cancro, "Cofinanziamento Ministero dell’Università e della Ricerca Scientifica e Tecnologica 1998", and Consiglio Nazionale delle Ricerche-targeted Project on Biotechnology (to G.C.), as well as by the Istituto Superiore di Sanità (Research Project "Artificial organs and organ transplantation" to G.C; "Pathology, clinic and therapy of AIDS" to G.C. and F.B.). I.S. is a Scholar of the Leukemia Society of America and was supported by National Institutes of Health Grants CA55735 and GM48614. Back

2 Address correspondence and reprint requests to Dr. Giovanni Camussi, Laboratorio di Immunopatologia Renale, Corso Dogliotti 14, 10126 Torino, Italy. E-mail address: Back

3 Abbreviations used in this paper: ft, fucosyltransferase; PI, propidium iodide; Rg, receptor globulin. Back

Received for publication November 19, 1998. Accepted for publication February 8, 1999.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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L. Biancone, V. Cantaluppi, D. Duo, M. C. Deregibus, C. Torre, and G. Camussi
Role of L-Selectin in the Vascular Homing of Peripheral Blood-Derived Endothelial Progenitor Cells
J. Immunol., October 15, 2004; 173(8): 5268 - 5274.
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